Technical Field
The present disclosure relates to compositions and methods for randomly introducing codon-mutations in a target nucleic acid molecule and, more particularly, using wild-type and triplet-randomized oligonucleotides to introduce mutations uniformly across a target nucleotide of interest in a controlled fashion and with a low rate of insertions or deletions.
Description of the Related Art
Libraries of mutant genes are generated for use in a wide-variety of biology and bioengineering applications, such as in directed evolution of enzymes and biotherapeutics (Cherry and Fidantsef, Curr. Opin. Biotechnol. 14:438, 2003; Jäckel et al., Annu. Rev. Biophys. 37:153, 2008), creation of computationally-focused protein libraries (Lutz and Patrick, Curr. Opin. Biotechnol. 15:291, 2004), stabilization of proteins (Eijsink et al., Biomol. Eng. 22:21, 2005), and examination of structure-function relationships by deep-sequencing (Araya and Fowler, Trends Biotechnol. 29:435, 2011). In essence, mutant libraries are useful for the study and engineering of any gene property that can be screened or selected for in a relatively high-throughput fashion.
Mutant libraries are considered most effective for these purposes when most variants are mutated at a modest level. For example, introduction of an average of between one and ten amino-acid mutations per gene (most typically around three) is preferred (Cirino et al., “Generating mutant libraries using error-prone PCR.” In Directed Evolution Library Creation, pp. 3-9, Humana Press, 2003) because this strikes a balance between introducing sufficient diversity and introducing too many mutations that may result in mostly inactive variants. The probability that a protein retains its proper fold declines exponentially with the number of mutations, so highly mutated variants are often nonfunctional (Drummond et al., J. Mol. Biol. 350:806, 2005).
But, the current techniques for creating such mutant libraries are limited. For example, existing techniques for mutating genes across their entire length rely on processes that introduce mutations at the nucleotide level, such as by error-prone PCR or chemical mutagenesis (Cirino et al., 2003). As a result, the only mutant codons that are introduced at an appreciable rate are those that differ from the wild-type codon by just a single-nucleotide. This is because nucleotide-level mutagenesis will only rarely mutate two adjacent nucleotides, and is even less likely to mutate all three nucleotides in a codon (see Miyazaki and Arnold, J. Mol. Evol. 49:716, 1999).
Another established technique does allow the introduction of mutations at the codon level, but only at a small number of pre-selected sites (Georgescu et al., “Saturation mutagenesis.” In Directed Evolution Library Creation, pp. 75-83, Humana Press, 2003). This technique uses PCR with oligonucleotides that have been randomized at triplet sites. If, for example, a triplet is randomized to NNN (wherein N is taken to denote any nucleotide), then all 64 codons will appear in the library at that one site. However, these methods lead to rapid accumulation of insertions and deletions (which generally inactivate a protein) as the number of mutated sites increase, rendering these methods ineffective for more than a few positions. Hence, such techniques can only be used to fully randomize one or a small number of codons.
In view of the limitations associated with the production of mutant libraries, there is a need in the art for alternative methods for creating mutant libraries in which codon mutations are introduced across the full length of a gene or other nucleic acid of interest at a controlled rate so that the typical clone will have a small number of codon mutations. The present disclosure meets such needs, and further provides other related advantages.